DRD2 Genotype and Prenatal Exposure to Tobacco Interact to Influence Infant Attention and Reactivity

The present study examined the effects of dopamine receptor D2 genotype and PTE status on early infant neurobehavior

Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Interacts with the Ku70 Subunit of the DNA-Dependent Protein Kinase Complex

<div><p>Deformed Epidermal Autoregulatory Factor 1 (DEAF1) is a transcription factor linked to suicide, cancer, autoimmune disorders and neural tube defects. To better understand the role of DEAF1 in protein interaction networks, a GST-DEAF1 fusion protein was used to isolate interacting proteins in mammalian cell lysates, and the XRCC6 (Ku70) and the XRCC5 (Ku80) subunits of DNA dependent protein kinase (DNA-PK) complex were identified by mass spectrometry, and the DNA-PK catalytic subunit was identified by immunoblotting. Interaction of DEAF1 with Ku70 and Ku80 was confirmed to occur within cells by co-immunoprecipitation of epitope-tagged proteins, and was mediated through interaction with the Ku70 subunit. Using <em>in vitro</em> GST-pulldowns, interaction between DEAF1 and the Ku70 subunit was mapped to the DEAF1 DNA binding domain and the C-terminal Bax-binding region of Ku70. In transfected cells, DEAF1 and Ku70 colocalized to the nucleus, but Ku70 could not relocalize a mutant cytoplasmic form of DEAF1 to the nucleus. Using an <em>in vitro</em> kinase assay, DEAF1 was phosphorylated by DNA-PK in a DNA-independent manner. Electrophoretic mobility shift assays showed that DEAF1 or Ku70/Ku80 did not interfere with the DNA binding of each other, but DNA containing DEAF1 binding sites inhibited the DEAF1-Ku70 interaction. The data demonstrates that DEAF1 can interact with the DNA-PK complex through interactions of its DNA binding domain with the carboxy-terminal region of Ku70 that contains the Bax binding domain, and that DEAF1 is a potential substrate for DNA-PK.</p> </div

DEAF1 interacts with the DNA-PK complex.

<p>Recombinant GST and GST-DEAF1 fusion proteins were isolated from bacterial extracts, bound to glutathione-Sepharose beads, and incubated overnight in the presence (+) or absence (−, buffer only) of PC-3 cell lysates. Interacting proteins were eluted, separated by SDS-PAGE, and stained with Coomassie blue (<b>A</b>) or analyzed by Western blot with an antibody to DNA-PKcs (<b>B</b>). The 70 kDa and 80 kDa proteins (arrows) were determined by mass spectrometry to be Ku70 and Ku80. (<b>C</b>) CV1 cells were transfected with expression plasmids for DEAF1-FLAG and/or HA-tagged Ku proteins. Cell lysates were immunoprecipitated (IP) with anti-FLAG coupled beads followed by Western blot analysis with anti-HA antibody. (<b>D</b>) Reversing the epitopes described in (C), lysates of cells transfected with DEAF1 and FLAG-tagged or HA-tagged Ku proteins were immunoprecipitated with anti-FLAG coupled beads followed by Western blot analysis with anti-DEAF1 antibody. Levels of the proteins in 1.0% of the cell lysates (inputs) used in the immunoprecipitation reactions in C and D were assessed using the antibodies indicated. The results are representative of two independent experiments.</p

Ku70 is unable to relocalize a cytoplasmic form of DEAF1 to the nucleus.

<p>CV-1 cells were transfected either alone or in combinations with DEAF1-HA, HA-Ku70, GFP-DEAF1, or GFP-DEAF1nls (mutated nuclear localization signal). Cellular localization of HA-tagged proteins were determined by indirect immunofluorescence using anti-HA antibodies and secondary antibodies conjugated to CY3 and GFP-tagged proteins were determined by intrinsic GFP fluorescence.</p

The DEAF1 DNA binding domain interacts with Ku70.

<p>(<b>A</b>) DEAF1 proteins with C-terminal and/or N-terminal deletions were radiolabeled with [³⁵S]methionine by <i>in vitro</i> translation and used in GST pull-downs. 10% of the [³⁵S]-labeled proteins (10% input) used in the pull-downs are shown to the left of the pull-down results obtained with GST-Ku70 and GST. Schematic representations of all the DEAF1 translated proteins tested are shown in the left panel. Results are summarized as a positive interaction (+) or no interaction (−). Ku80 interaction with GST-Ku70 was used as a positive control. (<b>B</b>) A schematic summary of the DEAF1 and Ku70 proteins and the interaction domains deduced from these experiments. Also shown are the Bax interaction domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033404#pone.0033404-Cohen1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033404#pone.0033404-Subramanian1" target="_blank">[24]</a>, the MYND (zinc binding) domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033404#pone.0033404-Lutterbach1" target="_blank">[25]</a>, and DBD (DNA binding domain).</p

DEAF1 interacts with Ku70.

<p>GST pull-downs assays were performed by incubation of <i>in vitro</i> translated, [³⁵S]methionine-labeled Ku70, Ku80, or DEAF1 with the indicated GST fusion proteins and GST. 10% of the <i>in vitro</i> translated proteins used in the pull-downs are shown on the left of each panel. The results are representative of two independent experiments.</p

DNA-PK phosphorylates DEAF1, and sequence specific DNA competes with Ku70 for binding to DEAF1.

<p>(<b>A</b>) DNA-PK and [γ-³²P]ATP were incubated with GST or GST-DEAF1 bound to glutathione-sepharose beads in the absence (−) or presence (+) of dsDNA (salmon sperm DNA sheared to ∼300 bp). Bound proteins were washed, eluted, and separated by SDS-PAGE; and the phosphorylated proteins were detected by autoradiography. Protein levels used in the reactions were assessed in separate SDS-PAGE gels stained with Coomassie blue. (<b>B</b>) Electrophoretic mobility shift assays were performed using the indicated FLAG proteins purified from HEK 293T/17 cells and either ³²P-labeled dsDNA probes N52-69 (DNA ligand of DEAF1) or Mut 2 (mutated DNA ligand that DEAF1 does not bind). The purities of the proteins used in the assays are shown in Coomassie blue stained SDS-PAGE gels on the left. (<b>C and D</b>) GST pull-down experiments using GST-Ku70 (550–609) or GST with <i>in vitro</i> translated, [³⁵S]methionine-labeled DEAF1 (167–565) were performed in the absence (−) or presence (+) of 15 µg of DEAF1 promoter DNA (<i>C</i>), or increasing amounts of N52-69 DNA (0.04 mg–0.6 mg) or 0.6 mg of Mut 2 DNA (<i>D</i>). 10% of the <i>in vitro</i> translated [³⁵S]methionine-labeled proteins used in each pull-down experiment are shown on the left. The results are representative of two independent experiments.</p

DEAF1 interacts with the C-terminal end of Ku70.

<p>(<b>A and B</b>) GST-Ku70 fusion proteins with N-terminal and/or C-terminal deletions of Ku70 were used in pull-downs with <i>in vitro</i> translated [³⁵S]methionine-labeled DEAF1 (167–565) shown in (A) or DEAF1 (155–326) shown in (B). The results are representative of two independent experiments. (<b>C</b>) GST tags were reversed relative to (A) and (B) and GST-DEAF1 was used to pull-down <i>in vitro</i> translated Ku70 peptides (right panel). A schematic representation of all the Ku70 translated proteins tested is shown in the left panel. Results are summarized as a positive interaction (+) or no interaction (−).</p

Gene–Environment Interactions across Development: Exploring DRD2 Genotype and Prenatal Smoking Effects on Self-Regulation

Genetic factors dynamically interact with both pre- and postnatal environmental influences to shape development. Considerable attention has been devoted to gene–environment interactions (G × E) on important outcomes (A. Caspi & T. E. Moffitt, 2006). It is also important to consider the possibility that these G × E effects may vary across development, particularly for constructs like self-regulation that emerge slowly, depend on brain regions that change qualitatively in different developmental periods, and thus may be manifested differently. To illustrate one approach to exploring such developmental patterns, the relation between variation in the TaqIA polymorphism, related to D2 dopamine receptor expression and availability, and prenatal exposure to tobacco was examined in two exploratory studies. First, in 4-week-old neonates, genotype–exposure interactions were observed for attention and irritable reactivity, but not for stress dysregulation. Second, in preschool children, genotype was related to Preschool Trail Making Test (K. A. Espy and M. F. Cwik, 2004) task performance on conditions requiring executive control; children with both the A1+ genotype and a history of prenatal tobacco exposure displayed disproportionately poor performance. Despite study limitations, these results illustrate the importance of examining the interplay between genetic and prenatal environmental factors across development